title: Tumor necrosis factor-α enhances RANKL expression in gingival epithelial cells via protein kinase A signaling.
abstract: OBJECTIVE
  Periodontitis is an inflammatory disorder of the supporting tissue of teeth, which is composed of gingival soft tissue, cementum covering the tooth root, alveolar bone and periodontal ligament. The receptor activator of nuclear factor kappa B ligand (RANKL) is known to be an essential factor for osteoclastogenesis. Recent clinical studies indicate that levels of RANKL in the gingival crevicular fluid are increased while levels of its decoy receptor, osteoprotegerin (OPG), are decreased in patients with periodontitis. Although the gingival sulcus is composed of gingival tissue, RANKL and OPG expression in gingival epithelial cells is not fully understood. The aim of this study is to investigate the expression of RANKL and OPG in gingival tissue and which factors regulate RANKL expression in gingival epithelial cells.
  
  
  METHODS
  Reverse transcriptase polymerase chain reaction analysis, western blotting and immunohistochemistry were performed to confirm RANKL and OPG expression in gingival epithelial cells (GECs) and in gingival tissue. Immunostaining was also examined to confirm tumor necrosis factor (TNF)-α and TNF receptor type 1 (TNFR1) expression in gingival tissue. Ca9-22 cells, a human gingival epithelial cell line and human primary GECs were treated with TNF-α. Ca9-22 cells were treated by antibodies against TNF receptors, an inhibitor and an activator of protein kinase A (PKA) signaling and inhibitors of p38, Erk and NF-κB signaling to examine TNF-α-RANKL signaling pathways.
  
  
  RESULTS
  RANKL mRNA and protein were expressed in GECs. Immunohistochemistry also showed RANKL expression in gingival tissue. On the other hand, the reverse transcriptase polymerase chain reaction and immunohistochemistry assay showed that GECs did not express OPG. In addition, TNF-α and TNFR1 proteins were expressed in junctional epithelium. TNF-α increased RANKL expression in GECs. TNF-α-induced RANKL expression was inhibited by an antibody against TNFR1 and an inhibitor of PKA signaling. Surprisingly, forskolin, a PKA activator, increased TNF-α-induced RANKL expression.
  
  
  CONCLUSIONS
  RANKL, TNF and TNFR1 were coexpressed in junctional epithelium of gingival tissue. TNF-α induced RANKL expression via TNFR1 and PKA signaling in GECs of junctional epithelium.
authors.affiliation: Department of Periodontology, Showa University School of Dentistry, Tokyo, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Fujihara
authors.forename: M
authors.initials: M
authors.lastname: Usui
authors.forename: G
authors.initials: G
authors.lastname: Yamamoto
authors.forename: K
authors.initials: K
authors.lastname: Nishii
authors.forename: Y
authors.initials: Y
authors.lastname: Tsukamoto
authors.forename: Y
authors.initials: Y
authors.lastname: Okamatsu
authors.forename: T
authors.initials: T
authors.lastname: Sato
authors.forename: Y
authors.initials: Y
authors.lastname: Asou
authors.forename: K
authors.initials: K
authors.lastname: Nakashima
authors.forename: M
authors.initials: M
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects*; D004846:Epithelial Attachment / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors; D005260:Female; D005881:Gingiva / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D008813:Mice, Inbred ICR; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D053244:Osteoprotegerin / Q000187:drug effects; D053245:RANK Ligand / Q000187:drug effects*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors
chemical_list: D016328:NF-kappa B; D053244:Osteoprotegerin; D053245:RANK Ligand; D047888:Receptors, Tumor Necrosis Factor, Type I; C506030:TNFRSF11B protein, human; C505688:TNFSF11 protein, human; D014409:Tumor Necrosis Factor-alpha; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases
keywords: gingival epithelial cell; protein kinase A signaling; receptor activator of nuclear factor kappa B ligand; tumor necrosis factor-α